Herein, methods for the synthesis of fatty acid esters (FAE) are described. Such methods are important in the analysis of fatty acids from foodstuffs and in the production of diesel fuel from biological sources (e.g., biodiesel or fatty acid methyl ester). While differing elements are of relevance to the disparate fields, both areas would benefit from a simple, robust method for the synthesis of FAE from biological materials.
The analysis of fatty acids has become increasingly important due largely to nutritional and health implications. Therefore, a method for analyzing fatty acids that provides rapid results without modification of the constituent fat compositions is of great value. Many methods are currently used to analyze fatty acids (e.g., Morrison and Smith, 1964; Sukhija and Palmquist, 1988; Kazala et al., 1999; Mir et al., 2002; Nuemberg et al., 2002; Budge and Iverson, 2003; Cooper et al., 2004). These methods, generally, are not convenient or direct, and often must be optimized for reaction conditions, including the catalyst and the temperature (Lewis et al., 2000; Park et al. 2002; Shahin et al., 2003). Ideal methods, as noted by Palmquist and Jenkins (2003) would determine the total fatty acid concentration in tissues, oils, and feed samples by converting fatty acid salts, as well as the acyl components in all lipid classes such as triacylglycerols, phospholipids, sphingolipids, and waxes, to FAE using a simple direct esterification procedure.
Current methods for FAE synthesis utilized for fat analysis require extensive sample pre treatment to eliminate the presence of water. Many of the common reagents employed within the esterification process are water sensitive (e.g. BF3, NaOCH3, NaOCH2CH3). Moreover, because the esterification reaction is an equilibrium wherein water is formed as a product, lower concentrations of water favor product formation. As a result, even within methods known to the art employing non water-sensitive reagents (e.g., H2SO4, HCl, NaOH), efforts to eliminate water from the reaction mixture are extensive and time consuming. These attributes of current methodologies underlie the pervasive recognition in the art that water within the reaction mixture decreases yield and reaction rates in FAE synthesis—thus water must be avoided in FAE synthesis. Within such methods, once the sample has been dried, incomplete dissolution &/or reconstitution of the sample within the water-free media also leads to decreased yields of FAE's due to the kinetic restrictions imposed on the system. Current methodologies employ extended reaction times to overcome such limitations, however as noted in the art extended reaction times also lead to increased degradation of polyunsaturated fats which alters the determination of a samples fat composition.
Likewise, the production of diesel fuel from biological sources is of growing interest. Generally, methodology employed in the synthesis of fatty acid esters (FAE) for biodiesel is similar to that employed within the analysis of fatty acids. A primary difference however is the focus on market-driven cost pressure, and the process cost is prominent in the practical production of biodiesel. The largest single cost of biodiesel production is the cost of the feedstock. Extensive extraction and processing of any oil-rich biomass is typically required and often employs extensive oil extraction (via oilseed crushing or solvent extraction with hexane or similar) and drying prior to the introduction of the oil into the fatty acid ester synthesis process. In addition to the concerns relating to the presence of water, yield of FAE and ensuring the final FAE product does not contain free fatty acid anion (FFAA) are of particular concern in the production of biodiesel. FFAA causes fuel combustion and corrosion problems and therefore is undesirable in fuel mixtures.
Considerations deriving from the costs and hazards of methods for oil extraction have led to a large body of work directed at minimization of FFAA concentrations and extraction process simplification. The former has focused primarily on FFAA in feedstocks (Hass et al., U.S. Pat. No. 6,399,800) and FFAA reprocessing (Stern et al. U.S. Pat. No. 5,424,466), while methods for FAE synthesis from low value, high FFAA concentrations feedstocks have been developed (Hass, et al. U.S. Pat. No. 6,855,838) in attempts to minimize feedstock costs. Additional methods for simplifying the extraction process (Hass et al. U.S. Pat. No. 7,612,221) for use in feedstocks with lipid-linked fatty acids have been developed.
Common to both fatty acid analysis and biofuel production, there is a pronounced need in the art for simple, rapid & robust methods for the dissolution and esterification of samples or feedstocks containing complex mixtures and/or heterogeneous structures.